The long-term objective of the proposed research is to elucidate the structural basis for the substrate specificities of cytochromes P450 2B from rat and human. For decades, these enzymes have served as a prototype for investigation of the mechanism by which drugs such as phenobarbital and environmental contaminants such as polychlorinated biphenyls activate gene expression. P450 2B enzymes are also very versatile catalysts with a broad range of substrates, including environmental toxicants, drugs, and steroids. Along with members of the P450 2A and 2C subfamilies, P450 2B enzymes exhibit the least degree of catalytic preservation across mammalian species. This observation suggests a strong role for steric constraints imposed by the enzymes, as opposed to the inherent chemical reactivity of the compounds, in determining substrate specificity. Through studies during the past decade the key amino acid residues that dictate ligand binding orientation within the interior of the active site and contribute to bioactivation and detoxification of a wide range of compounds have been identified. Such information has made it possible to explain many of the functional differences among P450 2B enzymes and to begin to alter their activities in a rational manner. Although more is known at present more about the structural determinants of P450 2B specificity than about any other mammalian subfamily, a number of important issues remain to be resolved. The central hypothesis of this proposal is that substrate specificity reflects the interplay between amino acid residues in the interior of the active site and those that line the substrate access channel. This hypothesis will be tested by a combination of site-directed mutagenesis, heterologous expression in E. coil, a variety of functional assays, 3D molecular modeling, and X-ray crystallography. The individual specific aims are to: 1) identify the substrate access channel in cytochrome P450 2B1 and determine the contribution to rates and regioselectivity of substrate hydroxylations; 2) rationally design cytochromes P450 2B with improved catalytic efficiency or novel substrate specificity; 3) determine the X-ray crystal structure of cytochrome P450 2B1. The results should provide new and important information about the specific interactions of cytochromes P450 with ligands and help improve safety assessment of chemicals and drugs and individual risk assessment upon exposure to xenobiotics.